P430: COMBINATORIAL ADAPTOR-MEDIATED TARGETING OF ACUTE MYELOID LEUKEMIA WITH CAR T-CELLS
Authors: Laura Volta, Renier Myburgh, Christian Pellegrino, Francesco Manfredi, Anne Kaiser, Monique Maurer, Jan Müller, C. Matthias Wilk, Marco Matteo Bühler, Chiara F Magnani, Dario Neri, Markus Manz
Journal: HemaSphere (2023)

Background: Chimeric Antigen Receptor (CAR) T-cells have been clinically implemented to eradicate B-cell malignancies by targeting lineage specific cell-of-origin antigens, eliminating both tumor cells as well as healthy B- and plasma-cell counterparts, which can subsequently regenerate again from hematopoietic stem and progenitor cells (HSPCs). Similar on-target, off-tumor side effects would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), where the heterogeneous overlapping antigen expression on tumor and HSPC would lead to terminal ablation of hematopoiesis. Thus, higher tumor-cell selectivity and/or tunable effector activity is required. Aims: Enhanced selectivity and on-demand activity can be achieved by Adaptor-mediated (Ad) CAR T-cell that do not directly recognize an antigen but bind to a specific tag placed on antigen-binding adaptors. Such tagged adaptors would then act as safety switch of the system, modulating CAR T-cell activation, proliferation and lytic activity. In addition, combinatorial use of multiple adaptors with different tumor-antigen specificities might enhance tumor selectivity. Methods: We engineered second generation CAR T-cells to display various anti-fluorescein binders, either in scFv format or with an artificial lipocalin scaffold. We further generated fluorescein-labelled adaptors in IgG or diabody format, directed against AML markers, determined by profiling patient-derived cells (i.e. CD117, CD33, CD371). Cytotoxicity elicited by adaptors were evaluated in vitro against various cell lines and primary patient samples. Pharmacokinetic studies were performed in NSG mice to determine serum half-life of adaptor molecules in vivo and AML-xenograft mouse models were generated to assess on-tumor residence time of the adaptors as well as the therapeutic efficacy when administered with AdCAR T-cells. Results: Site-specific conjugations of diabodies mediated the strongest AML killing over a broad range of concentrations, both against AML cell lines and primary blasts. Importantly, combinatorial use of adaptors led to higher adaptor-antigen recognition by CAR T-cells and resulted in significantly enhanced tumor cell lysis compared to equimolar concentrations of single adaptor in vitro. Anti-CD117 and anti-CD33 diabodies in combination with AdFITC CAR T-cells led to bone marrow (BM) elimination of leukemia cells in a therapeutic xenograft model. Importantly, in vivo imaging as well as terminal analysis showed that AdFITC CAR T-cells in combination with adaptors were equally efficient as direct CAR T-cells. Summary/Conclusion: We here provide proof-of-concept that AdFITC CAR T-cells and selective combinations of small antibody fragments as adaptors can be an efficient strategy for next-generation immuno-targeting, potentially allowing for individualized treatment and conditional cytotoxic activity, with the aim to recovesssr healthy HSPCs or permit subsequent HSC transplantation. Keywords: Immunotherapy, Cancer immunotherapy, AML, CAR-T